This invention relates to a method and formulation for creatine kinase separation by electrophoresis. More specifically, the present invention relates to a method and formulation for separation of creatine kinase isoforms by electrophoresis.
Creatine kinase or CK is a known enzyme, and CK activity has been found in several human tissues, the major sources being skeletal muscles, myocardium and brain. There are two creatine kinase subunits, M and B, which combine to form the three CK isoenzymes which may be found in human serum. These isoenzymes are dimers composed of the two subunits and have been identified as CK-BB, CK-MB and CK-MM. These three isoenzymes have also been referred to as CK-1, CK-2 and CK-3 based upon their relative degrees of movement during electrophoretic separation, CK-1 being the most anodal, CK-2 being less anodal and CK-3 being the least anodal. The CK-1 or CK-BB isoenzyme is found predominantly but not exclusively in brain tissue, the CK-3 or CK-MM isoenzyme is found predominantly but not exclusively in skeletal muscle, and the CK-2 or CK-MB isoenzyme is found predominantly but not exclusively in the myocardium.
CK isoenzyme testing, typically performed by electrophoresis, is a specific and reliable procedure frequently used to aid in the diagnosis of myocardial infarction.
Isoforms, also known as subtypes, subforms or isomers, are variants of CK-MM and CK-MB isoenzymes with different isoelectric points. Prior to the present invention, several investigators have reported that the CK-3 or CK-MM isoenzyme can be further broken down into as many as five isoforms which can be found by isoelectric focusing or column chromatofocusing techniques. CK-3 or CK-MM has been shown to be a dimer of two identical peptide chains containing a carboxy terminal lysine which serves as a substrate for serum carboxypeptidase. This particular chemical composition comprises one of the three major isoforms of CK-3. The removal of a lysine by a serum carboxypeptidase creates a more anodal migrating band during electrophoresis and comprises the second major isoform. The third isoform occurs when the terminal lysine is removed from the second peptide chain. This third isoform band migrates even more anodally than the other forms.
Utilizing the nomenclature system wherein the most anodal band is given the numeral 1, then among the isoforms of CK-MM (CK-3), the most anodal isoform would be CK-3.sub.1, the next most anodal would be CK-3.sub.2, and the next most anodal would be CK-3.sub.3, etc. The clinical significance appears to be, at the present time, that the actual length of time that CK has been released into the serum may be estimated by comparing the relative amounts of the CK isoforms present. Thus, the CK-3.sub.3 to CK-3.sub.1 ratio analysis gives an earlier indication of acute myocardial infarction then the mere presence of CK-2 heretofore relied upon. The ratio analysis has also been used during investigational or research procedures to monitor the effect of thrombolytic therapy and to document the time of myocardial occlusion onset more accurately.
Prior to the present invention it has also been reported that CK-2 or CK-MB is a dimer of one M subunit and one B subunit which can be further broken down into two isoforms where only the M subunit contains a carboxy terminal lysine. The removal of the lysine by a serum carboxypeptidase creates a more anodal migrating band during electrophoretic separation. Again using the nomenclature system where the most anodal band is given the numeral 1, then the isoforms of CK-MB (CK-2) would be identified as CK-2.sub.1 and CK-2.sub.2, with CK-2.sub.1 being the more anodal.
Prior to the present invention, however, CK-MM isoform separation has been obtained primarily by isoelectric focusing, by column chromatography or by cellulose acetate electrophoresis all of which are time consuming such that the results are not usually available to permit treatment on a real time basis. Furthermore, prior to the present invention, CK-MB isoform separation had been achieved primarily by isoelectric focussing or by column chromatography.